TCA Cycle Lecture 20 PDF

The TCA cycle The Krebs Cycle / The Citric Acid Cycle 4BICH001W Biochemistry Dr Sarah K Coleman  Previously… Glycolysis breakdown of GLUCOSE Has a net yield of 2x ATP, 2x NADH and 2x pyruvate molecules Most of the glycolysis enzymes are reversible, three are...

The TCA cycle The Krebs Cycle / The Citric Acid Cycle 4BICH001W Biochemistry Dr Sarah K Coleman  Previously… Glycolysis breakdown of GLUCOSE Has a net yield of 2x ATP, 2x NADH and 2x pyruvate molecules Most of the glycolysis enzymes are reversible, three are not Aerobically pyruvate converted to Acetyl CoA and enters the TCA cycle. Anaerobically it is fermented to lactate or ethanol to regenerate NAD+. Gluconeogenesis uses the reversible enzymes of glycolysis and different enzymes for the irreversible steps. Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  The TCA cycle the Tri-Carboxylic Acid cycle the Krebs cycle the Citric Acid cycle A central and fundamental metabolic pathway  Triacyl glycerols Polysaccharides sugars Proteins Glycerol Free fatty acids Beta oxidation Amino acid skeletons TCA Cycle Final common pathway for ‘fuel’ molecules Enter as Acetyl-CoA Provides energy transfer molecules Provide intermediate molecules for biosynthesis (anabolism) Occurs in mitochondria  The TCA Cycle The Krebs cycle, the Tri-carboxylic acid cycle (the TCA cycle) and the Citric Acid cycle all refer to the same thing. The cycle is named after Sir Hans Krebs. The cycle contains the six-carbon tricarboxylic acid, citric acid (citrate) as the first formed component. Occurs in mitochondrial matrix Sir Hans Krebs (1900-1981)  Brief History 1784 Citrate was discovered in lemon juice. 1900- 1920s Citrate shown to be widespread. Demonstrated that minced muscle tissue could catalyse the transfer of hydrogen atoms (a proton + an electron) from some organic acids: succinate, malate and citrate Shown by colour change of the redox dye methylene blue. The enzymes involved were termed dehydrogenases.  Methylene Blue Dye redox reaction Colour change 1930’s Measurement of oxygen uptake rate (usage) by the minced tissues showed that the acids were being rapidly oxidised to carbon dioxide. 1930’s Szent-Györgyi (Hungary) assembled these observations into the sequence of: Succinate – Fumarate – Malate – Oxaloacetate Albert Szent-Györgyi: The Nobel Prize in Physiology or Medicine 1937 "for his discoveries in connection with the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid" https://www.nobelprize.org/prizes/medicine/1937/szent-gyorgyi/facts/ Carl Martius and Franz Knoop showed that there was another sequence of: Citrate – Isocitrate – Ketoglutarate – Succinate  1937 Hans Krebs realised that these sequences were linked together as a cycle of reactions. Showed formation of citrate from oxaloacetate and pyruvate acetate There are very few cycles in biology, and he was awarded the Nobel prize for this insight. Hans Krebs : The Nobel Prize in Physiology or Medicine 1953 "for his discovery of the citric acid cycle" https://www.nobelprize.org/prizes/medicine/1953/krebs/facts/ The History of the Tricarboxylic Acid Cycle, Perspect. Biol. Med., 14: 154-170 (1970). Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). TCA Cycle Occurs in mitochondrial matrix in eukaryotes Some enzymes associated with inner membrane Glycolysis occurs where?  Roles of TCA cycle Final oxidative step in the catabolism of carbohydrates, fatty acids and amino acids. Provides a flow of simple carbon compounds into anabolic processes. Functions as a major source of energy, generating some ATP and a lot of NADH. (The NADH can be oxidised in the respiratory chain to generate more ATP, see next lecture)  Link with Glycolysis End product of glycolysis is the 3 carbon compound pyruvate. Pyruvate dehydrogenase converts pyruvate, CoA and NAD+ into Acetyl-CoA, CO2 and NADH. Acetyl-CoA feeds the 2 carbon acetyl group into the TCA cycle. Pyruvate dehydrogenase is a large, complicated molecule. It is a complex consisting of 3 distinct enzymes and needs 5 coenzymes. Fritz Lipmann - The Nobel Prize in Physiology or Medicine 1953 "for his discovery of co-enzyme A and its importance for intermediary metabolism“ https://www.nobelprize.org/prizes/medicine/1953/lipmann/facts/  Pyruvate Dehydrogenase (PDH) Catalyses pyruvate (3C) to acetyl-CoA (2C) Generates NADH CH3-CO-COO- + CoASH + NAD+ >>> CH3-CO-SCoA + CO2 + NADH Irreversible step – so key checkpoint in metabolism Enzyme complex inhibited by products; acetyl-CoA and NADH Also inhibited by high [GTP] and stimulated by high [AMP] Regulated by reversible phosphorylation (adding / removing a specific phosphate group). Done by other enzymes high ATP/ADP ratio > triggers phosphorylation > inhibits PDH Insulin triggers dephosphorylation > stimulates PDH  Pyruvate Dehydrogenase (PDH) Location: Mitochondria matrix (as are TCA cycle enzymes) Glycolysis occurs in cytosol. How does pyruvate pass through the mitochondrial membranes? Through outer membrane via large non-specific anion channels Through inner membrane via the selective transport protein complex Mitochondrial Pyruvate Carrier (MPC)  Summary of the cycle There are 8 different reaction steps: 1) Condensation of acetyl CoA (2C) with oxaloacetate (4C) to form citrate (6C). Enzyme is citrate synthetase. 2) Formation of isocitrate (6C) [via cis-aconitate], OH group moved. Enzyme is aconitase. 3) Oxidative decarboxylation of isocitrate (6C) to ketoglutarate (5C) and CO2. Enzyme is isocitrate dehydrogenase. 4) Oxidative decarboxylation of ketoglutarate (5C) to succinyl-CoA (4C) and CO2. Enzyme is α-ketoglutarate dehydrogenase.  Summary of the cycle 5) Conversion of succinyl-CoA (4C) to succinate (4C). Involves hydrolysis and phosphorylation. Enzyme is succinyl CoA synthetase. 6) Oxidation of succinate (4C) to fumarate (4C). Removal of 2 H to leave a double bond. Enzyme is succinate dehydrogenase. 7) Hydration of fumarate (4C) to malate (4C). Enzyme is fumarase. 8) Oxidation of malate to oxaloacetate. Enzyme is malate dehydrogenase. Why the cycle for oxidising acetate? Higher energy yield (eventually) Regenerates carrier, oxaloacetate Control of cycle Some enzymes inhibited by high [ATP] Some enzymes inhibited by high [product] Some enzymes stimulated by high [ADP] There are 3 key check points  The Checkpoint enzymes Citrate synthase (step 1) is allosterically inhibited by high [ATP]. (Km for acetyl-CoA is increased). ΔG°’ = -31.5 kJ/mol Isocitrate dehydrogenase (step 3) is inhibited by ATP and NADH; is stimulated by ADP. ΔG°’ = -21 kJ/mol α-ketoglutarate dehydrogenase (step 4) is inhibited by NADH and succinyl-CoA, and ATP. ΔG°’ = -33 kJ/mol The rate of the TCA cycle is reduced if ATP levels are high  Need for Oxygen… Krebs cycle does not use oxygen directly! Krebs cycle generates NADH and FADH2 which feed into Electron Transport Chain (ETC). Oxygen is used in Electron Transport Chain as ultimate electron acceptor NADH and FADH2 are RE-OXIDISED back to NAD+ and FAD in ETC so can be used again in Krebs cycle. Remember OILRIG  The Cofactors These are GTP (or ATP) and NADH, FADH2 (reduced form) NADH generated at steps 3, 4 and 8 Step 6 generates FADH2 Step 5 generates GTP (or ATP) for one turn of the cycle. Regeneration of these cofactors (to oxidised form): NAD+ and FAD are regenerated by the Electron Transport Chain. GDP can be regenerated by transferring a phosphate group from GTP to ADP by nucleoside diphosphate kinase. GTP + ADP -----> GDP + ATP Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Lipid metabolism and TCA cycle Fatty acid chains are broken down by beta-oxidation into 2C fragments This 2C fragment is acetyl-CoA. Acetyl-CoA feeds into the TCA cycle. Biosynthesis of fatty acids is reversal of this breakdown process. (Will learn more about this in level 5 Metabolic Biochemistry)  Protein metabolism and TCA cycle Proteins are broken down to amino acids. Amino acids undergo transamination and deamination reactions These generate TCA cycle intermediates. – E.g. aspartate has a structure very similar to oxaloacetate. Valine and methionine feed in as succinate. For biosynthesis of amino acids - important precursors are oxaloacetate and α-ketoglutarate. (Will learn more about this in level 5 Metabolic Biochemistry)  For biosynthesis of amino acids - important precursors are oxaloacetate and α-ketoglutarate. Amphibolic Reactions of TCA cycle Green arrows indicate replenishing intermediates for Kreb’s cycle (anaplerotic reactions) Red arrows indicate intermediates removed for biosynthesis (cataplerotic reactions)  Anaplerotic reactions If Krebs cycle intermediates are used up for biosynthesis, they need to be replenished to enable the cycle to continue. Anaplerotic (filling up) reactions are used to replace them. An example: pyruvate can be carboxylated to oxaloacetate by pyruvate carboxylase with CO2 and ATP. – (instead of being broken down to acetyl-CoA) This oxaloacetate is used to replenish any intermediates drawn off for biosynthesis. Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). Location of Glycolysis, TCA Cycle and Oxidative Phosphorylation in a Eukaryotic Cell  The yield of ATP from glucose breakdown… How much ATP is released from a molecule of glucose is dependent on: Oxidation of NADH and FADH2 in Electron Transport Chain. If no intermediates are taken off for biosynthesis from Krebs cycle. Problem: Inner mitochondrial membrane is impermeable to NADH. NADH generated in the cytoplasm cannot enter the mitochondrion to reach the respiratory chain. Location of Glycolysis, TCA Cycle and Oxidative Phosphorylation in a Eukaryotic Cell  The yield of ATP from glucose breakdown… Problem: Inner mitochondrial membrane is impermeable to NADH. Answer: The electrons carried by NADH are transported to the respiratory chain but not the NADH itself. There can be an energetic price to pay for the transport.  Two shuttles for ‘NADH electron’ transport Glycerophosphate shuttle (used by skeletal muscle) Cytosolic NADH (from glycolysis) passes its electrons across the membrane via glycerol-3-phosphate. Forms FADH2 (not NADH) inside mitochondrial matrix. This reduces ATP yield. Malate-Aspartate shuttle (used by liver, kidney, heart) Malate is used to carry electrons across inner membrane into matrix of mitochondrion. Generates NADH (and oxaloacetate) using malate dehydrogenase. Yield of ATP from oxidation of one molecule of glucose… So the maximal ATP yield will depend on the shuttle used to transport the electrons across the membrane. Let us assume 1 molecule of NADH generates 2.5 ATP and 1 molecule of FADH2 generates 1.5 ATP. Numbers used here will be explained in ETC and oxidative phosphorylation lecture.  Substrate Level Phosphorylation If the glycerol-3 phosphate shuttle used - gives FADH2 - makes 3 ATP (not 5) Why not concerned with shuttle here? Substrate Level Phosphorylation NADH gives 2.5 ATP and FADH2 gives 1.5 ATP  So, in summary the TCA cycle is… Final oxidative step in the catabolism of carbohydrates, fatty acids and amino acids. It provides a flow of simple carbon compounds into anabolic processes. It functions as a major source of energy compounds: One turn of cycle generating 1 ATP by SLP, 3 NADH (7.5 ATP) and 1 FADH2 (1.5 ATP). Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). MCQ quiz for Lecture 20: The TCA cycle Answers will be given in your Seminar sessions – with further discussion. You must attempt before your seminar session. These quizzes are part of your learning for the Biochemistry module They will aid your on-going studies at the University of Westminster  Q1) The TCA cycle takes place in which cellular compartment? A. Cytosol B. Mitochondrial inner membrane C. Mitochondrial intermembrane space D. Mitochondrial matrix E. All of the above Q2) Which of the following occur in the TCA cycle? A. Two carbons enter the cycle in the form of acetyl-CoA B. Oxidation of carbon compounds to produce CO2 C. Reduction of NAD+ and FAD D. Biosynthetic intermediates are produced E. All of the above Q3) How is the TCA cycle linked to glycolysis? A. In the absence of oxygen, pyruvate is converted to lactic acid which enters the TCA cycle B. Pyruvate is converted to CO2, NADH and acetyl-CoA, which enters the TCA cycle C. These processes are totally independent and are not linked D. The TCA cycle provides substrates for glycolysis E. The same enzymes are involved in both pathways  Q4) Which of the following are mechanisms by which enzymes of the TCA cycle are regulated? A. Inhibition by high [ATP] B. Stimulation by high [ADP] C. Inhibition by high [product] D. All of the above E. Unlike glycolysis, the TCA cycle is not regulated, the reactions proceed in a smooth cycle whenever there are available substrates  Q5) The TCA cycle is a process in which the metabolism of which molecules intersect? 1) Sugars A. 1, 2 and 4 2) Amino acids B. 1, 2, 3 and 5 3) Ethanol C. 2, 4 and 6 4) Fatty acids D. 3, 4, 5 and 6 5) Lactic acid E. 1, 2, 3, 4, 5 and 6 6) Prosthetic groups containing metal ions, such as haem